PROJECT SUMMARY Membraneless organelles have important functions in cellular physiology and pathology. Recent studies show that these organelles are formed through liquid-liquid phase separation of intrinsically disordered proteins (IDPs) and RNA molecules. IDPs phase separate into liquid droplets in test tubes and form P bodies or stress granules in stressed cells. Both mutant and wild type forms of several IDPs are found aggregated in neurons and associated with neurodegenerative disorders. However, very little is known about how IDPs misfold and aggregate in these organelles and how this process can be regulated. Lack of this knowledge is attributed to the current method that is used to monitor membraneless organelles in live cells: this process is visualized through imaging fluorescent protein-tagged IDPs to analyze changes of their location and diffusion rate before and after organelle formation. Nonetheless, this method does not reveal whether IDPs misfold or aggregate within the organelle, because the morphology remains unchanged before and after IDPs aggregation. To overcome this challenge, the PI has developed a novel imaging method, hereinafter named AggTag (aggregation tag), to enable fluorogenic detection (turn-on fluorescence) of misfolded soluble oligomers both in test tubes and live cells. In this MIRA proposal, the PI plans to further develop the AggTag method with new probes that can distinguish soluble oligomers from insoluble aggregates using orthogonal fluorescent signals (Project 1). This unprecedented resolution will allow the PI to ask how IDPs misfold and aggregate in phase separated droplets. The PI have begun this direction with a focus on a group of intrinsically disordered RNA binding proteins (RBPs), which harbor RNA binding domains (RBD) and disordered prion-like domains (PLD). While PLD has been the primary focus in literatures, preliminary data have led to a novel hypothesis that whether RBD misfolds contributes to whether RBP misfolds during and after formation of droplets. This hypothesis will be tested both in vitro and in live cells, using a combination of the AggTag method and biochemical assays (Project 2). Finally, the PI will develop chemical strategies to control phase separation and membraneless organelles. Although LLPS can be prevented and dissolved by small molecules, disruption of the liquid droplets could obstruct their physiological functions. Till now, no small molecules have been discovered to promote formation of liquid droplets and prevent RBP misfolding. Preliminary data indicate that sugar phosphates are a novel class of molecules that promote droplet formation, stabilize liquid droplets, and prevent RBP misfolding in droplets. Based on these results, the PI will use efforts from multiple disciplines to understand the mechanisms underlying the observed effects of sugar phosphates and further develop them into a class of chemical regulators with proper selectivity and efficacy (Project 3). In summary, the proposed research will provide an enabling technology to visualize misfolding and aggregation of proteins in membraneless organelles and generate novel chemical compounds to regulate this disease-related process.